Nasal Carriage of Methicillin-Resistant Staphylococcus Sciuri Group by Residents of an Urban Informal Settlement in Kenya

Background: The Staphylococcus sciuri group constitutes animal-associated bacteria but can comprise up to 4% of coagulase-negative staphylococci isolated from human clinical samples. They are reservoirs of resistance genes that are transferable to Staphylococcus aureus but their distribution in communities in sub-Saharan Africa is unknown despite the clinical importance of methicillin-resistant S. aureus. Objectives: We characterised methicillin-resistant S. sciuri group isolates from nasal swabs of presumably healthy people living in an informal settlement in Nairobi to identify their resistance patterns, and carriage of two methicillin resistance genes. Method: Presumptive methicillin-resistant S. sciuri group were isolated from HardyCHROM™ methicillin-resistant S. aureus media. Isolate identification and antibiotic susceptibility testing were done using the VITEK®2 Compact. DNA was extracted using the ISOLATE II genomic kit and polymerase chain reaction used to detect mecA and mecC genes. Results: Of 37 presumptive isolates, 43% (16/37) were methicillin-resistant including - S. sciuri (50%; 8/16), S. lentus (31%; 5/16) and S. vitulinus (19%; 3/16). All isolates were susceptible to ciprofloxacin, gentamycin, levofloxacin, moxifloxacin, nitrofurantoin and tigecycline. Resistance was observed to clindamycin (63%), tetracycline (56%), erythromycin (56%), sulfamethoxazole/trimethoprim (25%), daptomycin (19%), rifampicin (13%), doxycycline, linezolid, and vancomycin (each 6%). Most isolates (88%; 14/16) were resistant to at least 2 antibiotic combinations, including methicillin. The mecA and mecC genes were identified in 75% and 50% of isolates, respectively. Conclusion: Colonizing S. sciuri group bacteria can carry resistance to methicillin and other therapeutic antibiotics. This highlights their potential to facilitate antimicrobial resistance transmission in community and hospital settings. Surveillance for emerging multidrug resistant strains should be considered in high transmission settings where human-animal interactions are prevalent. Our study scope precluded identifying other molecular determinants for all the observed resistance phenotypes. Larger studies that address the prevalence and risk factors for colonization with S. sciuri group and adopt a one health approach can complement the surveillance efforts.


INTRODUCTION
T he Staphylococcus sciuri group (S. sciuri, S. lentus, and S. vitulinus) consists of coagulase-negative staphylococci that are distinguishable from other staphylococci by a positive oxidase test. 1 These bacteria can be isolated from the environment, animals, and dairy products. People may be colonized in the nasopharynx and skin 2 following repeated contact with colonized livestock and pets 2 or through contact with food animal products. 3 Nevertheless, human infection with the S. sciuri group does occur; they can constitute up to 4% of coagulase-negative staphylococci isolated from clinical samples, and can cause endocarditis, peritonitis, septic shock, urinary tract infections, pelvic inflammatory disease, and wound infections. 1 Over the last decade, oxacillin/ methicillin-resistant staphylococcal strains have emerged, increasing the medical relevance of the S. sciuri group. 3 These bacteria can receive 5 and transfer resistance genes to human and animal pathogens such as Staphylococcus aureus 4 and can carry a mecA gene that is closely related to the methicillin-resistant S. aureus (MRSA) mecA gene. 4 The mecA gene encodes broad-spectrum beta-lactam resistance. 4 A novel mecA homolog -mecC -which also confers resistance to methicillin, has emerged in staphylococci isolated from animals, humans, and the environment. 6 We characterised S. sciuri group isolates from nasal swabs collected from presumably healthy people to determine their antibiotic resistance profiles and the proportion harbouring mecA and mecC genes.

MATERIALS AND METHODS
During a 2019 population-based study on antimicrobial resistance in communities and hospitals in Kenya (KNH/UoN ERC# P164/03/2018), which targeted colonizing MRSA strains from presumably healthy people in Kibera-an informal settlement in Nairobiwe unexpectedly cultured S. sciuri group bacteria on HardyCHROM™ MRSA media (Hardy diagnostics, CA)chromogenic media that can isolate and differentiate S. aureus and other staphylococci. 7 Nasal swabs were plated on HardyCHROM™ MRSA media, followed by incubation at 37°C overnight (18-24 hrs). After incubation, single small blue colonies, presumptively identified as methicillin-resistant S. sciuri group, were collected from each positive agar plate and sub-cultured on tryptic soy agar plates (KEMRI Production Department, Nairobi), then incubated overnight.
DNA extraction from confirmed isolates was done using the ISOLATE II genomic kit (Bioline, FL) following manufacturer instructions, and stored at -20 o C until tested. The presence of mecA and mecC genes was determined using the S. aureus mecA and mecC primers on the VeritiPro Thermal Cycler (Thermo Fisher scientific, MA). Separate reaction mixes were prepared for mecA and mecC. Each 25 µL reaction mix consisted of 0.

Ethical Approval
The isolates analysed in this study were identified during sample processing for the ARCH study. The ARCH study was approved by the KNH/UoN ERC (# P164/03/2018).

DISCUSSION
Focus on the S. sciuri group bacteria has increased in recent years owing to their implication in opportunistic human and veterinary infections 9,10 food contamination, 11,12 and potential for zoonotic transmission. 13 These bacteria are natural reservoirs of methicillin resistance genes, which can be transferred to S. aureus-an important human and animal pathogen-and can carry virulence genes that promote pathogenicity in coagulase-negative staphylococci. 10,14 Colonising strains of methicillin-resistant S. sciuri group in the nasal cavities of presumptively healthy individuals in sub-Saharan Africa have not widely been reported. Consequently, little is known about the distribution of these bacteria within communities, despite their potential to transfer resistance genes to pathogenic staphylococci. Previous studies have reported low prevalence (~5%) of S. sciuri infections in hospitals-presumably crosstransmitted between patients and healthcare workers 15and in communities, presumably transmitted via close contact with animals. 2 It is likely that colonizing strains, as those found in our study, are transmitted via bioaerosols, with dust mediating the transfer of environmental bacteria when inhaled. 16 Informal settlements are commonly characterized by poor environmental hygiene, which can facilitate the thriving of S. sciuri group bacteria. Data from the parent study indicate that 81% of sampled households keep domestic animals, the majority (76%) of which are dogs 17 -a known reservoir of S. sciuri.
The susceptibility of S. sciuri group isolates to ciprofloxacin, gentamycin, levofloxacin, moxifloxacin, nitrofurantoin and tigecycline is consistent with another study. 15 This suggests that these antibiotics can be used in the management of infections caused by S. sciuri group bacteria. Conversely, we identified an isolate that was resistant to eight antibiotics, including vancomycin-used to treat S. sciuri infections 16 -highlighting the potential public health threat that can arise if such strains become amplified in communities and hospitals. While the clinical significance of S. sciuri group may be unappreciated, the capacity of these bacteria to carry multidrug resistance is well established 2,18 and has been reported in clinical studies in Serbia 3,15 and Nigeria. 19 The observed distribution of mecA and mecC genes in our sample is consistent with findings from a study in Tunisia. 13 Resistance to clindamycin can be mediated via the erm gene, which is located on transposon Tn554, which has insertion sites in the Staphylococcus spp. chromosome, where mecA and mecC genes are contained, 20 and may explain the observed resistance to clindamycin among the isolates with both mecA and mecC genes. Tetracycline resistance was common among isolates with the mecA gene, as demonstrated by other studies, 21 suggesting that tet and mecA genes may be located on the same genetic element. The absence of mecA and mecC genes in one isolate despite its resistance to clindamycin, daptomycin and methicillin suggests that other resistance elements mediate resistance to beta-lactams and other antibiotics within the group of bacteria. 13,22 One limitation of this study was its limited scope which precluded identifying other molecular determinants for the observed resistance phenotypes. Larger studies that address the prevalence and risk factors for colonization with S. sciuri group and adopt a one health approach can complement surveillance efforts.

CONCLUSION
Nasal colonization with methicillin-resistant S. sciuri group bacteria appears low in the population studied and may not be mediated by companion animals (e.g., dogs) which were prevalent in this population. Nevertheless, these bacteria are resistant to medically important antibiotics and carry important resistance genes, presenting a potential AMR threat.